[0001] Title of Invention
[0002]HEPATITE C VIRUS ENVELOPE TWO PROTEIN (E2) WICH LACKS ALL
[0003]OR PART OF THE HYPERVARIABLE REGION ONE (HVR1) ,
[0004]CORRESPONDING NUCLEIC ACIDS, CHIMERIC VIRUSES AND USES
[0007]The present invention relates to nucleic acid molecules that encode a hepatitis C virus (HCV) envelope two protein which lacks all or part of the hypervariable region one (HVR1) of the envelope two (E2) protein. The invention further relates to the use of the nucleic acid molecules and their encoded polypeptides as vaccine candidates.
[0008]Background of Invention
[0009]Hepatitis C virus (HCV) is a positive-sense single- strand RNA virus belonging to the Flaviviridae family of viruses (Rice, 1996).
[0010]At present, more than 80% of individuals infected with HCV become chronically infected and these chronically infected individuals have a relatively high risk of developing chronic hepatitis, liver cirrhosis and hepatocellular carcinoma (Hoofnagle, 1997). The only effective therapy for chronic hepatitis C, interferon (IFN) , alone or in combination with ribavirin, induces a sustained response in less than 50% of treated patients (Davis et al., 1998; McHutchinson et al . , 1998) . Consequently, HCV is currently the most common cause of end-stage liver failure and the reason for about 30% of liver transplants performed in the U.S. (Hoofnagle, 1997). As a result of the inability to develop a universally effective therapy against HCV infection, it is estimated that there are still more than 25,000 new infections yearly in the U.S. (Alter
1997) Moreover, since there is no vaccine for HCV, HCV remains a serious public health problem.
[0011]A remarkable characteristic of HCV is its genetic heterogeneity, which is manifested throughout the genome (Bukh et al . , 1995). HCV circulates as a quasispecies of closely related genomes in an infected individual. Globally, six major HCV genotypes (genotypes 1-6) and multiple subtypes (a, b, c, etc.) have been identified (Bukh et al . , 1993; Simmonds et al., 1993) . The nucleotide and deduced amino acid sequences among isolates within a quasispecies generally differ by < 2%, whereas those between isolates of different genotypes vary by as much as 35%.
[0012]The most heterogeneous regions of the genome are found in the two envelope genes El and E2; in particular, the hypervariable region 1 (HVRl) at the N-terminus of E2 (Hijikata et al . , 1991; einer et al . , 1991).
[0013]The fact that the HVRl is the region of the genome with the highest degree of genetic variability suggests that it is under strong immune pressure. Indeed, Ray et al. (1999) recently reported that patients who developed a chronic infection had a higher rate of non-synonymous mutations within the HVRl as compared with the El protein while the reverse was observed in patients who were able to clear the infection. These authors therefore hypothesized that the HVRl region of HCV might act as a decoy antigen by stimulating a strong immune response that is ineffective in clearing viremia.
[0014]However, the proposed role of the HVRl as an immunologic decoy is not easily reconciled with prior studies in which a hyperimmune rabbit serum raised against the HVRl was demonstrated to be capable of neutralizing HCV in vi tro (Farci et al . , 1996, Shimizu et al . , 1996). Moreover, the presence of amino acids in the carboxy-terminal half of HVRl which are
conserved across genotypes suggests that HVRl might be required for HCV replication.
[0015]Summary of Invention
[0016]The present invention relates to nucleic acid molecules which lack all or part of the coding sequence of the HVRl region of the envelope 2 (E2) gene of hepatitis C virus (HCV) .
[0017]In the first embodiment, the nucleic acid molecule of the invention comprises the genome of an infectious hepatitis C virus in which the HVRl of the envelope 2 gene of the infectious HCV has been deleted.
[0018]It is therefore an object of the invention to provide nucleic acid sequence which encodes infectious hepatitis C virus lacking HVRl. Such nucleic acid sequence is referred to throughout the application as "ΔHVRl-infectious nucleic acid sequence . "
[0019]In a second embodiment, the nucleic acid molecule comprises chimeric genomes of chimeric hepatitis C viruses in which the structural region (core and envelope genes) or the envelope gene of a pestivirus genome, [for example, a bovine viral diarrhea virus (BVDV) ] or a flavivirus genome (for example, a dengue virus or a yellow fever virus) are replaced by the corresponding structural region or El and E2 genes of an HCV in which the HVRl region of HCV E2 has been removed. It is therefore an object of the invention to provide nucleic acid sequence which encodes chimeric HCV lacking the HVRl of the HCV E2 gene. Such nucleic acid sequence is referred to throughout the application as "ΔHVRl-chimeric nucleic acid sequences."
[0020]The present invention also relates to the in vitro and in vivo production of ΔHVRl-infectious HCV or ΔHVRl-chimeric HCV
viruses from the ΔHVRl-infectious or ΔHVRl-chimeric nucleic acid sequences of the invention.
[0021]The present invention also relates to the use of the ΔHVRl viruses of the invention to identify cell lines capable of supporting the replication of the viruses.
[0022]The invention also relates to the use of the ΔHVRl infectious or chimeric nucleic acid sequences of the invention in the production of ΔHVRl-infectious HCV or ΔHVRl-chimeric HCV respectively, and the use of these virions for the development of inactivated or attenuated vaccines to prevent HCV in a mammal .
[0023]In a third embodiment, the DNA construct comprises an
[0024]HCV E2 gene lacking the HVRl region. Such a "ΔHVR1-E2 gene" may also be linked in tandem in the DNA construct with an HCV El gene. When contained alone or in tandem with the El gene, the ΔHVR1-E2 gene may be further modified at its carboxy-terminus to produce either a secreted or surface expressed ΔHVR1-E2 protein.
[0025]The invention further relates to pharmaceutical compositions and DNA-based vaccines which comprise the nucleic acid molecules of the invention.
[0026]The invention also relates to methods of preventing or treating HCV in a mammal comprising administering the nucleic acid molecules of the invention to a mammal in an amount effective to stimulate the production of a protective humoral and/or cellular immune response to HCV.
[0027]The invention also provides a kit for the treatment or prevention of HCV, the kit comprising a DNA molecule of the invention useful as an immunogen in generating a protective immune response to HCV.
[0028]The invention further relates to the use of the nucleic acid molecules of the invention as immunogens to
generate antibodies to the ΔHVRl-infectious HCV, the ΔHVRl- chimeric HCV or the ΔHVR1-E2 protein, preferably neutralizing antibodies. The invention therefore relates to the use of such antibodies in passive immunoprophylaxis and to pharmaceutical compositions which comprise these antibodies.
[0029]The invention also relates to transformation of host cells with nucleic acid molecules of the invention to produce host cells which express ΔHVRl-infectious HCV, the ΔHVRl- chimeric HCV or the ΔHVR1-E2 protein.
[0030]The invention further relates to the use of host cells expressing ΔHVRl-infectious HCV, the ΔHVRl-chimeric HCV or the ΔHVR1-E2 protein as immunogens to stimulate a protective immune response to HCV.
[0031]The present invention, of course, also relates to ΔHVRl E2 protein produced from the ΔHVR1-E2 gene constructs of the invention or obtained from the ΔHVRl-infectious HCVs or ΔHVRl-chimeric HCVs of the invention. These ΔHVR1-E2 proteins may be used as vaccines for immunizing mammals, especially humans, against HCV.
[0032]Brief Description Of Figures
[0033]Figures LA-IF show the nucleotide sequence (SEQ ID NO: 1) of the infectious hepatitis C virus clone of genotype la [H77C (ΔHVRl) ] which lacks the hypervariable region one (HVRl) of the second envelope protein and Figures 1G-1H show the amino acid sequence (SEQ ID NO: 2) encoded by the clone. The complete sequence of H77C (ΔHVRl) is identical to pCV-H77C but lacks the fragment from nucleotide positions 1491 to 1571 which encodes HVRl (Yanagi et al . , 1997; ATCC accession number PTA-157).
Figure 2 shows in vi tro transcription-translation of constructs E1E2-715 (Lanes 1, 3, 4, 5, and 6) and ΔHVR1-7 (Lane 8) with rabbit reticulocyte lysates with and without the addition of canine microsomal membranes.
[0034]Figure 3 shows the results of qualitative reverse transcriptase-nested polymerase chain reaction (RT-PCR) for HCV- RNA, logio HCV GE (genome equivalent) titer (in-house RT-PCR and Amplicor HCV Monitor, Roche Diagnostics) , second generation ELISA for anti-HCV and serum levels of alanine aminotransferase (ALT) in chimpanzee 1590 following transfection with RNA transcripts of the HCV deletion mutant H77C (ΔHVRl).
[0035]Figure 4 shows infection of chimpanzee 96A008 with HCV lacking HVRl. Serum samples were collected weekly from the chimpanzee and monitored for HCV-RNA [in-house RT-nested PCR and HCV Monitor test version 2.0 (Roche)], HCV antibodies (second generation ELISA, Abbott Laboratories) and liver enzyme levels (ALT, Anilytics) . PBMC were collected weekly and tested for HCV-specific proliferative capacity (peripheral CD4) with a panel of recombinant HCV proteins [C22 (core), C33-c (NS3), CIOO (NS3-NS4), and NS5] . The peripheral CD8+ T cell response (peripheral CTL) was tested by stimulating PBMC with a large panel of HCV peptides corresponding to known CTL epitopes. Expanded T cells isolated from liver biopsy samples were tested for HCV-specific proliferative responses (Intrahepatic CD4) +, positive; - negative. Liver biopsies were examined also for necroinflammatory changes [0 (normal) , 1+, 2+, 3+, 4+] . At week 0, the chimpanzee was inoculated intravenously with 90 ml of plasma from chimpanzee 1590 (week 4 after transfection) . At a titer of 10 GE/ml this represented an inoculum of approximately 900 genome equivalents of HCV.
Detailed Description of The Invention
[0036]The present inventors surprisingly observed that HCV cDNA from which the HVRl of the E2 gene had been deleted, encoded a hepatitis C virus which was able to replicate in vivo and stimulated a strong cellular immune response. Figure 1A-1F shows the nucleic acid sequence of this infectious HCV clone which was constructed using pCV-H77C (ATCC accession number PTA- 157), an infectious HCV clone of genotype la.
[0037]The present invention therefore relates in one embodiment to a nucleic acid molecule which comprises the genome of an infectious hepatitis C virus in which the HVRl of the envelope 2 gene of the infectious HCV has been deleted.
[0038]The E2 protein consists of amino acid 384 to 746 of the HCV polyprotein and the HVRl of the E2 protein consists of amino acid 384 to 410.
[0039]It is therefore an object of the invention to provide nucleic acid sequence which encodes infectious hepatitis C virus lacking HVRl. Such nucleic acid sequence is referred to throughout the application as "ΔHVRl-infectious nucleic acid sequence. "
[0040]It is understood that the deletion of HVRl of the E2 gene in the HCV genome can be made in infectious HCV clones of any genotype. For example, infectious HCV clones of different genotypes which have been constructed include those of Kolykhalov et al., (1997) and Yanagi et al. (1997, 1998) [who reported the derivation from HCV strains H77 (genotype la) and HC-J4 (genotype lb) of cDNA clones of HCV that are infectious for chimpanzees], and Yanagi et al. (Yanagi, 1999) [who reported the construction of an infectious cDNA clone from HCV strain HC- J6 (genotype 2a) ] . Preferably, the infectious HCV clones are infectious HCV clones of genotype la (ATCC accession number PTA-
157), lb (ATCC accession number 209596) or 2a (ATCC accession number PTA-153) .
[0041]In a second embodiment, the nucleic acid molecule of the invention comprises the genome of a flavivirus or pestivirus in which one envelope gene of the flavivirus or pestivirus is replaced by the E2 gene of an infectious HCV from which the HVRl region of HCV E2 has been removed.
[0042]It is therefore an object of the invention to provide nucleic acid sequence which encodes chimeric HCV lacking the HVRl of the HCV E2 gene. Such nucleic acid sequence is referred to throughout the application as "ΔHVRl-chimeric nucleic acid sequence. "
[0043]The Flaviviridae family of viruses which may be used to make the ΔHVRl-chimeric nucleic acid sequences include, but are not limited to, dengue virus, bovine viral diarrhea virus, yellow fever virus and Kunjin virus..
[0044]Preferably, the ΔHVRl-chimeric nucleic acid sequences of the invention are made using the structural region or El and E2 genes of an infectious HCV clone of any genotype. Preferably, the infectious HCV clones are infectious HCV clones of genotype la (ATCC accession number PTA-157), lb (ATCC accession number 209596) or 2a (ATCC accession number PTA-153) .
[0045]The deletion of HVRl which may be made in the sequence of the invention is at least 5 amino acids in length, preferably 10 amino acids, and most preferably the entire 27 amino acids of HVRl. The nucleic acid molecules of the invention therefore comprise genomes of HCV which lack at least a fragment of the HVRl sequence.
[0046]Of course, it is understood that in deleting all or part of the HVRl sequence, one may extend the deletions further in the carboxy-terminal direction of the E2 protein as long as
the resultant nucleic acid molecule is capable of replicating in a chimpazee and producing the E2 protein lacking all or part of HVRl.
[0047]The ΔHVRl-infectious nucleic acid sequence of the invention may further include one or more mutations such as those described in Example 4 which result in amino acid changes. One mutation is located within the E2 gene and results in a change from leucine to histidine at amino acid position 615. Another mutation is located within the NS3 serine-protease domain and results in a change from arginine to histidine at amino acid position 1143. A third mutation is located within the NS5B RNA-polymerase domain and results in a change from glutamic acid to aspartic acid at amino acid position 2875.
[0048]The present invention further relates to the in vitro and in vivo production of hepatitis C viruses from the ΔHVR1- infectious nucleic acid sequences of the invention, and the production of chimeric viruses from the ΔHVRl-chimeric nucleic acid sequences of the invention.
[0049]In one embodiment, the ΔHVRl-infectious nucleic acid sequences or the ΔHVRl-chimeric nucleic acid sequences of the invention can be inserted into an expression vector that functions in eukaryotic cells. Eukaryotic expression vectors suitable for producing high efficiency gene transfer in vivo are well known to those of ordinary skill in the art and include, but are not limited to, plasmids, vaccinia viruses, retroviruses, adenoviruses and adeno-associated viruses.
[0050]The sequences contained in the recombinant expression vector can then be transcribed in vitro by methods known to those of ordinary skill in the art in order to produce RNA transcripts which encode the HCV or chimeric viruses of the invention. The HCV or chimeric viruses of the invention may
then be produced by transfecting cells by methods known to those of ordinary skill in the art with either the in vitro transcription mixture containing the RNA transcripts or with the recombinant expression vectors containing the nucleic acid sequences described herein.
[0051]Where transfection of cells with recombinant expression vectors containing the nucleic acid sequences of the invention is used, transfection may be done by methods known in the art such as electroporation, precipitation with DEAE-Dextran or calcium phosphate, or incorporation into liposomes.
[0052]Suitable cells or cell lines for culturing the HCV or chimeric viruses of the invention include, but are not limited to, EBTr and Huh7.
[0053]The present invention also relates to the use of the ΔHVRl-infectious nucleic acid sequences or the ΔHVRl-chimeric nucleic acid sequences of the invention to identify cell lines capable of supporting the replication of HCV and the chimeric viruses of the invention.
[0054]The invention further relates to the use of the ΔHVRl- infectious nucleic acid sequences or the ΔHVRl-chimeric nucleic acid sequences of the invention to develop inactivated or attenuated vaccines to prevent hepatitis C in a mammal. For example, virions from cell lines infected with the HCV or chimeric viruses of the invention, or transfected with a ΔHVRl- infectious nucleic acid sequence or a ΔHVRl-chimeric nucleic acid sequence of the invention, can be purified from the cells and inactivated by methods known to those of ordinary skill in the art. The inactivated virions can be used to immunize mice, and if neutralizing antibody to HCV is produced, the virions can then be used to immunize chimpanzees to determine whether the antibodies are protective. Alternatively, cells infected with
the viruses of the invention may be passaged in cell culture to produce attenuated viruses which can be tested as candidate live vaccines. In assaying the ability of the viruses of the invention to infect mammals one can assay sera or liver of the infected mammal by RT-PCR to determine viral titer. In addition, the virulence phenotype of the virus produced by transfection of mammals with the sequences of the invention can be monitored by methods known in the art such as measurement of liver enzyme levels (alanine aminotransferase (ALT) or isocitrate dehydrogenase (ICD) ) or by histopathology of liver biopsies .
[0055]When used as a vaccine, the HCV or chimeric virions can be administered alone or in a suitable diluent, including, but not limited to, water, saline, or some type of buffered medium. The vaccine according to the present invention may be administered to an animal, especially a mammal, and most especially a human, by a variety of routes, including, but not limited to, intradermally, intramuscularly, subcutaneously, or in any combination thereof. Of course, it is understood that formulations or compositions comprising the HCV or chimeric virions of the invention may be used either therapeutically or prophylactically to treat or prevent the signs and symptoms of Hepatitis C.
[0056]In a third embodiment, the nucleic acid molecule of the invention comprises an HCV E2 gene lacking the HVRl region. Such a "ΔHVR1-E2 gene" may also be linked in tandem in the DNA construct with an HCV El gene.
[0057]When contained alone or in tandem with the El gene, the ΔHVR1-E2 gene may be further modified at its carboxy- terminus to produce either a secreted or surface expressed ΔHVR1-E2 protein.
A ΔHVR1-E2 gene encoding E2 protein targeted to the cell surface is preferred. Such a construct may be constructed by fusing an endoplasmic reticulum signal sequence to the amino- terminus of the nucleic acid sequence which encodes the truncated E2 gene fused at its carboxy-terminus to a plasma membrane anchor sequence.
[0058]By endoplasmic reticulum (ER) signal sequence is meant a nucleic acid sequence which encodes a continuous stretch of amino acids, typically about 15 to about 25 residues in length, which are known in the art to be generally located at the amino terminus of proteins and are capable of targeting proteins to the endoplasmic reticulum. Such ER signal sequences are known to those of skill in the art (see, for example, van Heijne, G. J. Mol. Biol., (1985) 184:99-105) and those of skill in the art would understand that even though their amino acid sequences may vary, such ER signal sequences are functionally interchangeable. Examples of ER signal sequences which may be used in the chimeric genes of the invention include, but are not limited to, the 20-carboxy-terminal amino acids of the full-length HCV El protein (amino acids 364-383 of the HCV polyprotein) , which serves as the natural signal sequence of the E2 protein or the urine Ig kappa-chain V-J2-C signal peptide sequence contained in the pDisplay vector.
[0059]Where the truncated HCV envelope protein is a truncated E2 protein, the approximately 30 carboxy-terminal amino acids of E2 have been identified to contain an ER retention sequence and its removal and replacement with a plasma membrane anchor sequence is believed to be critical for expression of the truncated E2 protein on the cell surface. Thus, the truncated E2 protein contains a truncation of at least the 20 carboxy-terminal amino acids of the full-length E2
protein, more preferably, a truncation of at least the 25 carboxy-terminal amino acids, and most preferably, a truncation of at least about the 30 carboxy-terminal amino acids. By "plasma membrane anchor sequence" as used in the chimeric gene of the invention is meant a nucleic acid sequence which encodes an amino acid sequence that allows for retention of at least part of the protein in the plasma membrane of a cell. At a minimum, a plasma membrane anchor sequence encodes a sequence of hydrophobic amino acids of sufficient length to span the lipid bilayer of the plasma membrane. Such hydrophobic sequences are known in the art as transmembrane domains and are typically found at the carboxy-terminus of many proteins found on the surface of cells or virions. These transmembrane domains are typically at least 20 to 30 amino acids in length and are followed by charged cytoplasmic domains of varying lengths.
[0060]It is therefore understood that the plasma membrane anchor sequence encoded by the coding sequence of the invention may contain in addition to a transmembrane domain of a virion or a protein found on the surface of a cell, a cytoplasmic domain.
[0061]Perferably, the encoded plasma membrane anchor sequence is at least twenty amino acids in length, more preferably, from about 20 to about 100 amino acids in length, and most preferably, from about 30 to about 70 amino acids in length. Examples of plasma membrane anchor sequences include, but are not limited to, hydrophobic transmembrane domains of receptors such as those for insulin and for a number of growth factors including platelet-derived growth factor (PDGF) and epidermal growth factor (EGF) , as well as the transmembrane domains of viral proteins that are anchored in the lipid envelope of the intact virion such as the transmembrane domains of the vesicular stomatitis and rabies virus G proteins.
Preferred plasma membrane anchor sequences for inclusion in the chimeric genes of the invention are sequences which encode the 50 amino acid transmembrane domain of the PDGF receptor as contained in the pDisplay vector described in the Examples, the - carboxy-terminal 64 and 37 amino acids respectively of the CD4 and decay accelerating factor (DAF) proteins (these sequences constitute the transmembrane and cytoplasmic domains of the CD4 and DAF proteins) and the 49 carboxy-terminal amino acids of the VSV G protein (also constituting the transmembrane and cytoplasmic domains of the VSV G protein) .
[0062]Of course, one of ordinary skill in the art would readily understand that other transmembrane domains suitable for use as plasma membrane anchor sequences in the chimeric genes of the invention are known or could be readily identified by carrying out carboxy-terminal deletions of known plasma membrane or viral envelope proteins (see, for example, Men et al (J. Virol. (1991) 65; 1400-1407).
[0063]A ΔHVR1-E2 gene encoding a secreted E2 protein may be constructed by fusing an endoplasmic reticulum signal sequence to the nucleic acid sequence which encodes the truncated E2 gene. In this case, the truncated E2 gene contains a truncation of at least the 31 carboxy-terminal amino acids (amino acids 716-746) , and more preferably, a truncation of at least the 85 carboxy-terminal amino acids (amino acids 662-746).
[0064]Like the ΔHVRl-infectious nucleic acid sequences and the ΔHVRl-chimeric nucleic acid sequences described above, it is understood that the ΔHVR1-E2 nucleic acid sequences of the invention may lack all or part of the HVRl sequence. The deletion of HVRl is at least 5 amino acids in length, preferably 10 amino acids, and most preferably the entire 27 amino acids of
HVRl. The ΔHVR1-E2 nucleic acid sequences of the invention may also contain further truncations at the carboxy-terminus of the E2 gene of HCV, and/or mutations at amino acid position 615 as described above.
[0065]The present invention therefore relates to insertion of the nucleic acid molecules comprising the ΔHVR1-E2 gene of the invention into a suitable expression vector that functions in eukaryotic cells, preferably in mammalian cells. By suitable it is meant that the vector is capable of carrying and expressing a chimeric gene of the invention. The expression vector therefore contains at least one promoter and any other sequences necessary or preferred for appropriate transcription and translation of the ΔHVR1-E2 gene. Preferred expression vectors include, but are not limited to, plasmid vectors.
[0066]The invention also relates to the use of expression vectors containing the ΔHVR1-E2 nucleic acid molecules of the invention as immunogens to produce protective antibodies to HCV. Direct transfer of the ΔHVR1-E2 nucleic acid sequences of the invention to a mammal, preferably a primate, more preferably a human, may be accomplished by injection by needle or by use of other DNA delivery devices such as the gene gun. Possible routes of administration of the expression vector include, but are not limited to, intravenous, intramuscular, intradermal, subcutaneous, intraperitoneal and intranasal.
[0067]Since the existence of different genotypes with a low degree of homology within the envelope proteins diminishes the hope of identifying conserved neutralization epitopes (Bukh J, et al, Se Liver Pis (1995); 15:41-63), it is likely that a polyvalent vaccine will be needed to generate broadly reactive neutralizing antibodies. Thus, in a preferred embodiment, nucleic acid molecules comprising ΔHVR1-E2 nucleic acid
sequences of isolates from multiple genotypes of HCV may be administered together to provide protection against challenge with multiple genotypes of HCV.
[0068]Accordingly, those of ordinary skill in the art would readily understand that multiple copies of different ΔHVR1-E2 nucleic acid sequences may be inserted into a single vector such that a host cell transformed or transfected with the vector will produce multiple envelope proteins. For example, a polycistronic vector in which multiple different ΔHVR1-E2 genes may be expressed from a single vector is created by placing expression of each gene under control of an internal ribosomal entry site (IRES) (Molla, a. et al. Nature, 356:255-257 (1992); Gong, S.K. et al. J. of Virol. , 263:1651-1660 (1989)). In a preferred embodiment, copies of different ΔHVR1-E2 nucleic acid sequences are inserted into multiple vectors and transformed or transfected into host cells so that multiple envelope proteins can be produced.
[0069]The expression vectors containing the ΔHVR1-E2 nucleic acid sequences of the invention may be supplied in the form of a kit, alone, or in the form of a pharmaceutical composition.
[0070]Suitable amounts of material to administer for prophylactic and therapeutic purposes will vary depending on the route selected and the immunogen (E2 gene with different deletions) administered. One skilled in the art will appreciate that the amounts to be administered for any particular treatment protocol can be readily determined without undue experimentation. The vaccines of the present invention may be administered once or periodically until a suitable titer of anti-HCV antibodies appear in the blood. A suitable amount of expression vector to be used for prophylactic purposes might be expected to fall in the range of from about 1 μg to about 5 mg,
more preferably from about 100 μg to about 5 mg, and most preferably from about 1 mg to about 2 mg. Such administration will, of course, occur prior to any sign of HCV infection. Further, one of skill in the art will readily understand that the amount of vector to be used will depend on the size and species of animal the vector is to be administered to.
[0071]A vaccine of the present invention may be employed in sterile liquid forms such as solutions or suspensions. Any inert carrier is preferably used, such as saline or phosphate- buffered saline, or any such carrier in which the expression vector of the present invention can be suitably suspended. The vaccines may be in the form of single dose preparations or in multi-dose flasks which can be utilized for mass-vaccination programs of both animals and humans. Of course, specific adjuvants such as CpG motifs (Krieg, A.K. et al.(1995) Nature 374:546 and Krieg et al . (1996)) J. Lab. Clin. Med. , 128:128) may prove useful with DNA-based vaccines or other vaccines .
[0072]The DNA-based vaccines will normally exist as physically discrete units suitable as a unitary dosage for animals, especially mammals, and most especially humans, wherein each unit will contain a predetermined quantity of active material calculated to produce the desired immunogenic effect in association with the required diluent. The dose of said vaccine or inoculum according to the present invention is administered at least once. In order to increase the antibody level, a second or booster dose may be administered at some time after the initial dose. The need for, and timing of, such booster dose will, of course, be determined within the sound judgment of the administrator of such vaccine or inoculum and according to sound principles well known in the art. For example, such booster dose could reasonably be expected to be advantageous at
some time between about 2 weeks to about 6 months following the initial vaccination. Subsequent doses may be administered as indicated.
[0073]The ΔHVR1-E2 nucleic acid sequences of the present invention can also be administered for purposes of therapy, where a mammal, especially a primate, and most especially a human, is already infected, as shown by well-known diagnostic measures. When expression vectors containing the chimeric genes of the present invention are used for such therapeutic purposes, much of the same criteria will apply as when they are used as a vaccine, except that inoculation will occur post-infection. Thus, when the expression vectors of the present invention are used as therapeutic agents in the treatment of infection, the therapeutic agent comprises a pharmaceutical composition containing a sufficient amount of the expression vector so as to elicit a therapeutically effective response in the organism to be treated. Of course, the amount of pharmaceutical composition to be administered will, as for vaccines, vary depending on the immunogen contained therein and on the route of administration.
[0074]The therapeutic agent according to the present invention can thus be administered by subcutaneous, intramuscular, intradermal or intranasal routes. One skilled in the art will certainly appreciate that the amounts to be administered for any particular treatment protocol can be readily determined without undue experimentation. Of course, the actual amounts will vary depending on the route of administration as well as the sex, age, and clinical status of the subject which, in the case of human patients, is to be determined with the sound judgment of the clinician.
[0075]The therapeutic agent of the present invention can be employed in sterile liquid forms such as solutions or
suspensions. An inert carrier is preferably used, such as saline, phosphate-buffered saline, or any such carrier in which the expression vectors of the present invention can be suitably suspended. The therapeutic agents may be in the form of single dose preparations or in multi-dose flasks, which can be utilized for mass-treatment programs of both animals and humans. Of course, when the expression vectors of the present invention are used as therapeutic agents, they may be administered as a single dose or as a series of doses, depending on the situation as determined by the person conducting the treatment.
[0076]The invention also relates to transformation of host cells with nucleic acid molecules of the invention to produce host cells which express HVRl-infectious HCV, the ΔHVRl-chimeric HCV or the ΔHVR1-E2 protein.
[0077]The invention further relates to the use of host cells expressing ΔHVRl-infectious HCV, the ΔHVRl-chimeric HCV or the ΔHVR1-E2 protein as immunogens to stimulate a protective immune response to HCV.
[0078]The present invention, of course, also relates to
[0079]ΔHVRl E2 protein produced from the ΔHVR1-E2 gene constructs of the invention or obtained from the ΔHVRl-infectious HCVs or ΔHVRl-chimeric HCVs of the invention.
[0080]The nucleic acids, polypeptides and viruses of the present invention can also be utilized in the production of antibodies against HCV. The term "antibody" is herein used to refer to immunoglobulin molecules and immunologically active portions of immunoglobulin molecules. Examples of antibody molecules are intact immunoglobulin molecules, substantially intact immunoglobulin molecules and portions of an immunoglobulin molecule, including those portions known in the
art as Fab, F(ab')2 and F(v) as well as chimeric antibody molecules .
[0081]Thus, the polypeptides, viruses and nucleic acid sequences of the present invention can be used in the generation of antibodies that immunoreact (i.e., specific binding between an antigenic determinant-containing molecule and a molecule containing an antibody combining site such as a whole antibody molecule or an active portion thereof) with antigenic determinants on the surface of hepatitis C virus particles.
[0082]The present invention therefore also relates to antibodies produced following immunization with the nucleic acid sequences, viruses or polypeptides of the present invention. These antibodies are typically produced by immunizing a mammal with an immunogen or vaccine to induce antibody molecules having immunospecificity for polypeptides or viruses produced in response to infection with the nucleic acid sequences of the present invention. When used in generating such antibodies, the nucleic acid sequences, viruses, or polypeptides of the present invention may be linked to some type of carrier molecule. The resulting antibody molecules are then collected from said mammal. Antibodies produced according to the present invention have the unique advantage of being generated in response to authentic, functional polypeptides produced according to the actual cloned HCV genome.
[0083]The antibody molecules of the present invention may be polyclonal or monoclonal. Monoclonal antibodies are readily produced by methods well known in the art. Portions of immunoglobin molecules, such as Fabs, as well as chimeric antibodies, may also be produced by methods well known to those of ordinary skill in the art of generating such antibodies.
The antibodies according to the present invention may also be contained in blood, plasma, serum, hybridoma supernatants, and the like. Alternatively, the antibody of the present invention is isolated to the extent desired by well known techniques such as, for example, using DEAE Sephadex. The antibodies produced according to the present invention may be further purified so as to obtain specific classes or subclasses of antibody such as IgM, IgG, IgA, and the like. Antibodies of the IgG class are preferred for purposes of passive protection.
[0084]The antibodies of the present invention are useful in the prevention and treatment of diseases caused by hepatitis C virus in animals, especially mammals, and most especially humans .
[0085]In providing the antibodies of the present invention to a recipient mammal, preferably a human, the dosage of administered antibodies will vary depending on such factors as the mammal's age, weight, height, sex, general medical condition, previous medical history, and the like.
[0086]In general, it will be advantageous to provide the recipient mammal with a dosage of antibodies in the range of from about 1 mg/kg body weight to about 10 mg/kg body weight of the mammal, although a lower or higher dose may be administered if found desirable. Such antibodies will normally be administered by intravenous or intramuscular route as an inoculum. The antibodies of the present invention are intended to be provided to the recipient subject in an amount sufficient to prevent, lessen or attenuate the severity, extent or duration of any existing infection.
[0087]The invention also provides that the nucleic acid molecules, viruses, polypeptides and antibodies of the invention may be supplied in the form of a kit, alone or in the form of a pharmaceutical composition.
All scientific publication and/or patents cited herein are specifically incorporated by reference. The following examples illustrate various aspects of the invention but are in no way intended to limit the scope thereof.
[0088]EXAMPLES Materials and Methods
[0089]Construction of An Expression Vector ΔHVR1-7 Containing El and a Carboxy-Terminal Truncated E2 Which Lacks the HVRl Region and the Carboxy-Terminal 31 Amino Acids of E2
[0090]Deletion of the nucleotide sequence encoding the HVRl region was performed by fusion PCR using pCV-H77C, the infectious cDNA clone of HCV genotype la (Yanagi et al . , 1997;ATCC accession number PTA-157) .
[0092]Primers used for the construction of ΔHVRl*
[0095]E2-Pstl: 5'-ACG CGT CTG CAG CTT AAT GGC CCA GGA CGC GAT GCT TG-3'
[0097]El-BglU: 5'- ACG CGT AGA TCT TAC CAA GTG CGC AAT TCC TCG GGG-3'
[0099]E1-383R (+10) : 5' -TCA GTT GGA TAG CGT CGA CGC CGG CAA ATA GCA G-3'
[0101]E2-411S (+10): 5'-CGT CGA CGC GAT CCA ACT GAT CAA CAC CAA CGG C-3'
[0103]HVRl-mutS: 5' -CTT GTA CCA TCA ATT ACA CCA TAT TC-3'
[0105]HVRl-mutR: 5' -GAT AGT GCC AAT GCC TAT ACG GG-3'
[0107]FUSION 1: 5' -CGT ATA GGC ATT GGC ACT ATC CTT GTA CCA TCA ATT ACA CC-3'
[0109]FUSION2: 5' -GGT GTA ATT GAT GGT ACA AGG ATA GTG CCA ATG CCT ATA CG-3'
[0111]E2-364 Notl: 5'-TTT TTT TTG CGG CCG CAT GGT GGG GAA CTG GGC GAA GGT CC-3'
[0113]E2-661-HindIII: 5' -ACG CGT AAG CTT CTA TTA CTC GGA CCT GTC CCT GTC TTC CAG-3'■^Restriction sites within primers are underlined
[0114]In the first step, the sequences encoding the El protein (aa 192-383) and a truncated E2 protein (aa 411-715) lacking the HVRl region (aa 384-410) were PCR amplified from pCV-H77C. Briefly, 50 ng of cDNA were added to a master mix containing 4 μl of 5X Advantage KlenTaq Buffer, 1.25 μl of dNTP (10 mM) , 1 μl of 10 μM sense primer [El-Bglll (SEQ ID NO: 4) for El and E2-411S(+10) (SEQ ID NO: 6) for E2], 1 μl of lOμM antisense primer [E1383R (+10) for El and E2-Pstl for E2] and 1 μl of the Advantage KlenTaq polymerase mixture. Cycling conditions were 99°C for 1 min followed by 25 cycles of 99°C for 35 sec, 67°C for 30 sec, 68°C for 3 min 30 sec. After purification of the PCR products, a fusion PCR was performed. Briefly, 2.5 μl of each PCR product were added to a master mix containing 10 μl of 10X Pfu buffer, 2 μl of 10 mM dNTP, 5 μl of 10 μM sense primer (El-Bglll) (SEQ ID NO: 4), 5 μl of 10 μM antisense primer (E2-Pstl) (SEQ ID NO: 3), 1 μl of Pfu and 72 μl of H20. Cycling conditions were 95 °C for 1 min, 67 °C for 1 min, 72 °C for 3 min 30 sec (30 cycles) . The fusion PCR product was digested with Bglll and Pstl and cloned into the expression vector pDisplay (Invitrogen) . A clone with an insert of the correct size (ΔHVR1-7) was selected and sequenced. Sequence analysis of both strands of DNA confirmed that this clone contained the expected sequence of El and a truncated E2 lacking the HVRl, i.e., ΔHVR1-7 encodes amino acids 192-383 of the El protein fused to amino acids 411-715 of the E2 protein.
[0115]Construction of An Infectious clone of HCV Lacking HVRl
[0116]Construction of an infectious cDNA clone that was full-length except for the HVRl was performed by digestion of the expression vector ΔHVR1-7 with Muni (which cuts at
nucleotide positions 1254 and 1983 of pCV-H77C) and cloning of the resulting fragment into the digested HCV cDNA clone H77C. Briefly, a 3 μg sample of the infectious HCV cDNA clone H77C was digested with Muni for 3 hours and the enzyme was then inactivated at 65 °C for 20 min. The digested cDNA clone, lacking a portion of the El and E2 regions (nucleotides 1255 to 1983 of pCV-H77C) , was dephosphorylated with calf intestinal alkaline phosphatase. A sample of ΔHVR1-7 was then digested with Muni for 3 hours. After inactivation of the Muni enzyme, the digested fragment was ligated into the digested pCV-H77C using standard procedures (Forns et al . , 1999). A clone containing the correct insert was selected, retransformed and large-scale plasmid DNA was prepared as previously described (Yanagi et al . , 1997). The complete sequence of the HVRl deletion mutant [H77C (ΔHVRl) ] was the expected one, that is, identical to pCV-H77C but lacking the fragment from nucleotide positions 1491 to 1571 which encodes HVRl.
[0117]In vi tro transcription-translation analysis
[0118]Non-linearized plasmid ΔHVR1-7 and the control plasmid
[0119]E1E2-715 were used for in vi tro transcription-translation. The
[0120]ΔHVR1-7 plasmid contained the nucleotide sequence encoding El
[0121](aa 192-383) and E2 (aa 411-715) inserted between a leader sequence which targeted the HCV proteins to the secretory pathway and the transmembrane domain of the platelet-derived growth factor receptor (PDGFR) which anchored the HCV E2 proteins to the plasma membrane in the expression vector pDisplay (Invitrogen) . The control plasmid contained the nucleotide sequence encoding El (aa 192-383) a carboxy-truncated form of E2 (aa 384-715) of pCV-H77C inserted between a leader sequence which targeted the HCV proteins to the secretory pathway and the transmembrane domain of PDGFR which anchored the
HCV E2 proteins to the plasma membrane in the expression vector pDisplay. Reactions were performed in 25 μl of the TNT Coupled Reticulocyte Lysate System (Promega) containing [S35] methionine, with or without the addition of canine microsomal membranes at 30 °C for 90 min. Total translation products were separated in 12% SDS/PAGE and identified by autoradiography .
[0122]Analysis of cell surface-expression of the E2 protein
[0123]Huh7 cells grown in 4-well tissue culture chambers were transfected with plasmids E1E2-715 and ΔHVR1-7 described above. Immuno-fluorescence analysis was performed 48 hours after transfection. Live cells were incubated for 30 minutes with a 1:100 dilution of rabbit hyperimmune serum ( Lmf86) raised against a peptide encoding the carboxy-terminal 21 amino acids of HVRl (aa 390-410 of pCV-H77C) or a rabbit hyperimmune serum { FOR-1 ) raised against a peptide within E2, but outside the HVRl (aa 517-535 of pCV-H77C) . After washing, cells were incubated with FITC-conjugated goat anti-rabbit IgG (1:100) for 30 min at 37 °C. After washing, slides were mounted and examined for immunofluorescence. For intracellular immunofluorescence staining , cells were fixed and permeabilized with cold acetone, as previously described (Forns et al . , 1999).
[0124]Transfection of a chimpanzee with H77C (ΔHVRl) RNA
[0125]RNA was transcribed in vi tro with T7 RNA polymerase from 10 μg of template plasmid H77C (ΔHVRl) linearized with Xbal as described previously (Yanagi et al., 1997). The quality and amount of RNA were analyzed by gel electrophoresis and ethidium bromide staining. Two transcription mixtures were each diluted with 400 μl of ice-cold phosphate-buffered saline without calcium or magnesium and then immediately frozen on dry ice and stored at -80°C. Within 24 hours, both transcription mixtures
were percutaneously injected into the liver of a chimpanzee (number 1590) under ultrasonographic control. The housing, maintenance, and care of the animals were in compliance with all relevant guidelines and requirements.
[0126]Serum samples were collected weekly from the chimpanzee and monitored for serum levels of alanine aminotransferase (ALT) , anti-HCV antibodies [second generation ELISA] and HCV-RNA [HCV Monitor test (Roche) and in-house RT- nested PCR (Bukh et al . , 1998)]. Sequence analysis of the recovered virus was performed at different time points during follow-up. In short, genomic regions were amplified in RT- nested PCR (Bukh et al . , 1998) with primers specific for the H77 strain of HCV.
[0127]Construction of vectors that Express Mutant E2 Protein on the Cell Surface or in a Form That Is Secreted
[0128]A. Cell Surface ΔHVR1-E2 Vectors
[0129]Clones E1E2-715 (amino acids 192-715) and ΔHVR1-7 (amino acids 192-383 of El in frame with amino acids 411-715 of E2) were produced as described above.
[0130]Clone ΔHVRl-mut5 (amino acids 192-383 of El in frame with amino acids 411-715 of E2 except that amino acid 615 is changed from a leucine to a histidine was produced as follows.
[0131]Primers El-Bglll (SEQ ID NO: 4) and HVRl-mutR (SEQ ID NO: 8) (containing a T->A mutation at nucleotide position 2185: see Example 4) were used to amplify a fragment encoding the El protein and a portion of the E2 protein using Advantage KlenTaq Polymerase mix, as described above. Primers HVR-1 mutS (SEQ ID NO: 7) and E2-Pstl (SEQ ID NO: 3) were used to amplify the rest of the truncated E2 protein by using AmpliTaq Gold DNA polymerase. In a second step, both products were fused by an
amplification reaction that contained a mixture of both external primers (El-Bglll (SEQ ID NO: 4) and E2-Pstl (SEQ ID NO: 3) and the two fusion primers SEQ ID NO: 9 and SEQ ID NO: 10) (Table 1), by using Advantage KlenTaq Polymerase mix. The fusion product was digested with Bglll and Pstl and cloned into the digested pDisplay vector to produce ΔHVRl-mut5.
[0132]Clones E1E2-715, ΔHVR1-7 and ΔHVRl-mut5 were used to transfect Huh7 cells and to determine their pattern of reactivity against a panel of 5 rabbit hyperimmune sera and 12 human monoclonal antibodies (Cardoso et al . , 1998, Inchauspe et al. 1998) by immunofluorescence in live and fixed/permeabilized cells .
[0133]B. Secreted ΔHVR1-E2 Vectors
[0134]In addition, three different expression vectors encoding a secreted form of the E2 protein were constructed: E2- 661, encoding the ER signal sequence of El (aa 364-383 of pCV- H77C) and a carboxy-terminal truncated E2 protein (aa 384-661 of pCV-H77C) ; E2-661 ΔHVRl, encoding the same protein with a deletion of the HVRl region (aa 384-410 of pCV-H77C) ; and E2- 661 ΔHVRl-mut, encoding the same protein with a deletion of the HVRl and the replacement of leucine for histidine at amino acid position 615. In this case, amplification was performed from templates E1E2-715, ΔHVR1-7 and ΔHVRl-mut5, respectively, with primers shown in Table 1. PCR products were cloned into the expression vector pcDNA3.1(-) (Invitrogen) . Clones E2-661, E2- 661 ΔHVRl, and E2-661 ΔHVRl-mut were used to express soluble E2 protein in vi tro . Briefly, one microgram of each of the plasmids was used for in vi tro transcription-translation. Reactions were performed in 25 μl of the TNT Coupled Reticulocyte Lysate System (Promega) containing [Ξ35] methionine,
with the addition of canine microsomal membranes at 30 °C for 90 min.
[0136]In vi tro transcription-translation analysis
[0137]To determine whether cleavage between El and E2 was affected by the deletion of the HVRl region, expression vectors E1E2-715 (encoding the complete El protein and a cell-surface E2 protein lacking the carboxy-terminal 31 amino acids of E2) and ΔHVR1-7 (encoding the complete El protein and a cell-surface E2 lacking the HVRl and carboxy-terminus 31 amino acids) were assayed for in vi tro protein synthesis. In vi tro transcription- translation was performed with the TNT Coupled Reticulolysate System using [S35] methionine . In the absence of microsomal membranes, translation products of the expected size were obtained for E1E2-715 and ΔHVR1-7 (Figure 2) . When microsomal membranes were added, cleavage between El and E2 was observed in both cases (Figure 2) and, as expected, the E2 protein derived from ΔHVR1-7 was slightly smaller in size. These results indicate that cleavage between El and E2 is not affected by the deletion of the N-terminus (HVRl) of the E2 protein.
[0139]Analysis of cell-surface expression of the E2 protein
[0140]To analyze whether a truncated E2 protein lacking the
[0141]HVRl region could be targeted to the cell surface, Huh7 cells were transfected with E1E2-715 and ΔHVR1-7. After transfection, fixed/permeabilized cells and live cells were analyzed for expression of E2 by immunofluorescence . Up to 20% of cells transfected with E1E2-715 were positive when stained with a rabbit hyperimmune serum lmf 86 or FOR-1 . Both fixed
/permeabilized and live cells were stained. A similar proportion of cells transfected with ΔHVR1-7 was positive when stained with rabbit hyperimmune serum FOR-1 (raised against aa 517-535) . Again, both fixed/permeabilized and live cells were stained. In contrast, there was no staining with rabbit hyperimmune serum lmf86, which was raised against aa 390-410 (within HVRl) . These results demonstrated that transport of the E2 protein to the cell surface was not affected by deletion of the HVRl region, suggesting that deletion of the N-terminal domain of E2 did not result in misfolding of this protein.
[0143]Transfection of a chimpanzee with RNA of H77C (ΔHVRl)
[0144]To determine the infectivity of H77C (ΔHVRl), RNA transcripts were injected into the liver of chimpanzee 1590. HCV-RNA was detected by RT-nested PCR at week one post- inoculation (p.i.) and the animal remained HCV-RNA positive throughout the entire follow-up period of 22 weeks (Figure 3) . The quantitative HCV Monitor test was negative until week 10 p.i. The genome titer during the first 7 weeks was D 10 genome equivalent (GE)/ml. However, beginning at week 8, the titer progressively increased over time (Table 2 and Figure 3) .
[0145]Table 2 Chimpanzee 1590 Infected with H77C (ΔHVRl)
[0146]Week HCV RNA* Titert MonitorΦ E2 NS3 NS5B in house (nt 2185) (nt 3769) (nt 8966)
[0156]9 + 2 - T/A G/a T/a
[0157]10 + 2 119 A/t G/a T/a
[0166]* In-house RT-PCR qualitative test t loglO HCV GE titer by in-house RT-PCR
[0167]Φ Amplicor HCV monitor (number of genome copies/ml) (Roche Diagnostics)
[0168]Nucleotide sequence corresponding to Yanagi 1997 Note: Lower case letters represent minor species
[0169]Serum liver enzyme values remained normal during the follow-up. The second generation anti-HCV test remained negative at least through week 22 p.i.
[0170]The follow-up period for chimpanzee 1590 has been extended to 72 weeks. Chimpanzee 1590 remained HCV-RNA positive throughout the 72 week follow-up period. Thus, the chimpanzee developed a chronic infection. In addition, the genome titer reached 103 - 104 GE/ml at weeks 11-14 (see Table 2) and remained at 103 - 104 GE/ml throughout the remainder of the 72 week period.
[0171]At weeks 51 and 52 p.i., the relative genome titer of HCV lacking HVRl was also assessed in serum, PMBC, and liver obtained from the transfected chimpanzee. HCV RNA was not
detected in PBMCs, but the HCV titers determined from liver tissue at weeks 51 and 52 p.i. were higher than the titers determined from the serum.
[0172]The 72 week follow-up period also revealed that the transfected chimpanzee became positive for antibodies to second generation ELISA HCV at week 37 and third generation RIBA (Chiron) confirmed the presence of antibodies to C22 (core) , C33- c (NS3), and clOO (NS3-NS-4).
[0173]Finally, CD4+ and CD8+ T cell responses were also measured. An HCV-specific proliferative CD4+ T cell response to C22 (core), C33-c (NS3) , clOO (NS3-NS4 ) and/or NS5 antigens of HCV was detected in the peripheral blood mononuclear cells (PBMC) beginning at week one post-infection and throughout the follow-up period. During most weeks, however, only a mono- specific response to the core was detected.
[0174]Peripheral CD8+ T cell (CTL) responses were tested at weeks 5, 9, 14, and 18 by an in vi tro peptide stimulation assay. CTL were detected with two NS4 peptides at weeks 9 and 14 and with one of these at week 18. A transient and monospecific (NS5) proliferative T cell response was detected in the liver at weeks 14 and 15. Although serum liver enzyme values remained normal during the entire follow-up, necroinflammatory changes indicative of hepatitis were detected in liver biopsies during weeks 34-40.
[0176]Analysis of the HCV-RNA recovered from chimpanzee 1590
[0177]Sequence analysis of the HCV-RNA recovered from chimpanzee 1590 was performed at week 13. The entire ORF was amplified by RT-nested PCR of overlapping gene regions. Direct sequence analysis of both strands of DNA from the PCR products
confirmed that the recovered virus lacked the HVRl region. Interestingly, three nucleotide mutations were identified; all three mutations were non-synonymous i.e. resulted in an amino acid change (Table 2) . One mutation was located within the E2 region (corresponding to aa position 615 of H77C) and generated a change from leucine to histidine. Another mutation was located within the serine-protease domain of NS3 (corresponding to aa position 1143 of H77C; arginine to histidine) . Both mutations are at positions that are universally conserved among all HCV genotype reference strains (Bukh et al . , 1998). Finally, a third mutation was observed in the NS5B RNA- polymerase domain (corresponding to aa position 2875 of H77C; glutamic acid to aspartic acid) . Although this is not a universally conserved position, a substitution of aspartic acid has not been described previously. Thus, all three mutations were unique.
[0178]Two additional mutations were identified upon rereading of the week 13 sequence: mutation at 514 (valine to methionine) and a synonymous mutation at amino acid 2631. Sequence analysis of the HCV-RNA recovered from chimpanzee 1590 was also performed at week 24 and five nucleotide mutations were identified; four of which were non-synonymous. (Table 3).
[0180]Evolution of HCV lacking HVRl m chimpanzee 1590' We performed sequence analysis of the entire open reading frame of the virus recovered from chimpanzee 1590 at weeks 13 and 24 post-transfection We confiπned that the HVRl region was not present and identified differences from the wild-type cDNA clone as indicated
[0182]'Nucleotide (nt) and amino acid (aa) positions of H77C Sequence of H77C(ΔHVR1) cDNA is shown on top Dominant sequences recovered from the chimpanzee are shown in capital letters Minor sequences recovered from the chimpanzee are shown in lower case leners tlogio titer determined by RT-nested PCR on 10-fold serially diluted extracted RNA Jlogio titer det ermmed by second generation Monitor test (Roche)
[0183]Four of the mutations were identical to the mutations identified at week 13 and one of the week 13 mutations (the NS3 mutation at amino acid 1143) reverted to wild-type. The single new mutation identified at week 24 was located within the serine protease domain of NS'3 (corresponding to aa position 1456 of H77C; threonine to methionine) .
[0184]To analyze a possible relationship between the increase in viral titer and the appearance of the four non- synonymous mutations identified at week 13, sequence analysis was performed from regions encompassing the described mutations. During weeks 4-7, direct sequence analysis did not reveal evidence of the mutated nucleotides at the four positions. Between weeks 8 and 14, coinciding with the first increase in viral titer, direct sequence analysis revealed the presence of amino acid substitutions at four positions (two in E2, one in NS3, and one in NS5B) (See Table 3) . Therefore, there was a temporal association between the appearance of these mutations and an increase in viral titer (See Tables 2 and 3) .
[0185]Example 5 Transmission of HCV lacking HVRl to a second chimpanzee
[0186]To investigate whether the HCV mutant lacking HVRl was transmissible, a naϊve chimpanzee (#96A008) was inoculated intravenously with 90 ml of plasma taken at week 4 from chimpanzee 1590, before appearance of consenus mutations in that chimpanzee (Table 3) . The virological and immunological correlates of HCV infection in chimpanzee 96A009 are shown in
[0187]Fig. 4. Serum HCV-RNA was first detected at week 2 p.i., with a genome titer of 101 GE/ml and the titer increased to peak levels of 10-105 GE/ml during weeks 3-9. The infection was resolved at week 18 p.i. Antibodies to HCV were not detected. A peripheral
mono-specific proliferative CD4+ T cell response against core (C22) was detected during weeks 4-13 p.i.; a multi-specific response against NS3 (C33-c) , NS3-NS4 (clOO) and NS5 was detected at week 18. The chimpanzee mounted an early and multi- specific peripheral CD8+ T cell response to a total of seven different epitopes (representing core, NS3, NS4 and NS5) at the weeks tested (weeks 4, 7, 11, 15 and 19) . Finally, a multi- specific (C33-c, clOO and NS5) sustained proliferative CD4+ T cell response was detected in the liver from week 8 p.i. Liver enzyme values were marginally elevated during weeks 10-18 p.i. and necroinflammatory changes were detected in liver biopsies during weeks 11-19 p.i.
[0188]Sequence analysis of the entire open reading frame of HCV recovered from the chimpanzee 96A008 at weeks 4 and 9 p.i. showed that the transmitted virus lacked HVRl. Compared with the cDNA clone of that mutant, four nucleotide substitutions were identified in the virus and all four mutations were non- synonymous. (Table 4) .
[0190]Evolution of HCV lacking HVRl in chimpanzee 96A008* We performed sequence analysis of the entire open reading frame of the virus recovered from chimpanzee 96A008 at weeks 4 and 9 post-inoculation We confirmed that the HVRl region was not present and identified differences from the wild-type cDNA clone as indicated
[0191]Week Titer Titer nl 1097 a. 252 In-houset Monitor} A K
[0193]2 1 Neg A T K/N A M G R C
[0194]3 3 3 33 T N A M G/a R/h C
[0195]4 4 4 00 T N A M G/a R/h C
[0200]9 5 4 62 T N G/A R/H C
[0212]•Nucleotide (nt) and am o acid (aa) positions of H77C Sequence of H77C(ΔHVR1) cDNA is shown on top Dominant sequences recovered from the chimpanzee are shown in capital letters Minor sequences recovered from the chimpanzee are shown in lower case letters tlogio titer determined by RT-nested PCR on 10-fold serially diluted extracted RNA {logio titer determined by second generation Monitor test (Roche)
[0213]Sequence analysis of regions encompassing the described mutations showed that a mutation in E2 and NS3 each had replaced the wild-type nucleotide by the time the virus was first detected (week 2 ) . An identical change in E2 had occurred in the transfected chimpanzee (Table 3 ) . A change in El appeared as the quasispeicies at week 2 but had replaced the wild-type nucleotide completely by week 3. The other mutation, in NS3 , appeared at week 3 and persisted as a quasispecies during weeks 3-10 and then reverted to wild-type . This latter mutation had been seen also in the transfected chimpanzee . (Table 3 ) .
Example 6
[0214]Analysis of potential conformational changes within E2 after L615H substitution
[0215]Sequence analysis of HCV recovered from chimpanzee 1590 demonstrated that a non-synonymous mutation occurred within the E2 protein during follow-up (nt 2186 of pCV-H77C; amino acid position 615) . Since a change from a non-polar amino acid
[0216](leucine) to a charged one (histidine) within E2 might change its conformation, Huh7 cells were transfected with E1E2-715
[0217](intact E1-E2), ΔHVR1-7 (the deletion of HVRl) or ΔHVRl-mut
[0218](deletion of HVRl along with the mutation at position 615) , and the transfected cells were stained for E2 with a panel of 4 rabbit hyperimmune sera and 12 human monoclonal antibodies
[0219](Cardoso et al . 1998; Inschauspe et al . , 1998). Immunofluoresecence analysis showed that there was no significant change in the patterns of reactivity among the three different forms of E2 targeted to the cell surface (Table 5) .
[0221]Effect of deletion of the HVRl region and mutation of E2 615 (leu-his) on the reactivity of cell surface-expressed E2
[0222]Antibody E1E2-715 ΔHVRl-7 ΔHVRl-mut5 RHS lmf86 (aa390-410) ++++ - - RHS 521 (aa 645-662) ++ +++ ++ RHS FOR-1 (aa 517-535) +++ +++ +++ RHS 554 (aa 460-483) ++++ +++ +++ 2H1 + /- - - 2G1 ++ ++ + 1H3 ++ +++ + 4F7 ++ ++ + 8E8 - - - 2H8 +++ +++ ++ 4E5 ++ +++ ++ 4F1 ++ +++ +++ 7B7 +++ ++ ++ 108F6 ++ + ++ 503E7p +++ ++ + 29E4p ++ ++ +
[0223]-RHS: rabbit hyperimmune sera.
[0224]-Amino acid position within the H77C sequence of the peptides used to generate RHS are indicated in parenthesis.
[0225]-The remaining 12 antibodies are human monoclonal antibodies (Cardoso et al . 1998; Inschauspe et al . , 1998) .
[0226]-Scoring system from - to ++++ . - means no staining and ++++ means strong staining.
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